Source: Schaaf, K., et al. A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis. J. Vis. Exp. (2017)
This video demonstrates an assay to evaluate the efficacy of drugs against Mycobacterium tuberculosis (Mtb) infection. An in vitro infection model containing Mtb/macrophage aggregates is generated that mimics in vivo infection. Upon introducing suitable antibiotics, their efficacy is tested via resuzarin assay to monitor the survival of bacteria with increasing drug concentrations.
1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc26206 (Mtb-GFP)
NOTE: The M. tuberculosis H37Rv derived auxotroph strain mc26206 (ΔpanCD, ΔleuCD) transformed with the gfp (green fluorescent protein) expressing plasmid pMN437 is used throughout this protocol. It is possible to substitute the Mtb-GFP strain with non-gfp expressing wild-type strain to enable easier accessibility of the strain for researchers. However, gfp expression is desirable to enable visual confirmation of phagocytosis and the formation of Mtb/macrophage aggregates. For long-term storage, Mtb-GFP were frozen at -80 °C in complete 7H9 media (described in step 1.1) supplemented with 20% glycerol.
2. Culture Conditions for THP-1 Cells
3. Infection Protocol to Generate Mtb/Macrophage Aggregate Structures
4. Growth Inhibition Assay to Assess Drug Efficacy Against Mtb Derived from Mtb/Macrophage Aggregates
5. Quantification of Drug Efficacy Using the Resazurin Microtiter Assay
Figure 1: Drug plate layout. (A) Example template of the drug challenge plate used in step 4. This allows for the parallel testing of two drugs in triplicate wells at 8 defined concentrations. As a representative experiment, we used rifampicin (RIF) and moxifloxacin (MOXI) starting at 2 µM and 2.5 µM, respectively. (B) An alternative template for throughput screening is also provided to show the possibility of testing of 58 compounds at a single concentration.
Figure 2: Generation of Mtb/macrophage aggregate structures in 96-well plate format. (A) Representative bright field and GFP merged images of Mtb-macrophage aggregates on Day 9 post-infection. Images were captured with a 4X objective using an automated cell imaging program that documents the entire well by stitching a montage of 3 by 3 individually captured images. (B) An individual non-stitched image from the visual field is shown in (A). (C) A representative merged bright field and GFP image of an Mtb/macrophage aggregate structure captured with a 10X objective.
The authors have nothing to disclose.
7H9 | BD Difco | 271310 | Follow manufacturer's recommendations |
Middlebrook OADC | BD Biosciences | 212351 | |
Tyloxapol | Sigma | T8761 | Prepare 20% stock solution in H2O; filter sterilize |
D-Pantothenic acid hemicalcium salt | Sigma | P5710 | Prepare 24 mg/ml stock solution in H2O; filter sterilize |
L-leucine | MP Biomedicals | 194694 | Prepare 50 mg/ml stock solution in H2O; filter sterilize |
Hygromycin B | EMD Millipore | 400051 | Prepare 200 mg/ml stock solution in H2O |
Nalgene Square PETG media bottle | Thermo Fisher | 2019-0030 | |
RPMI 1640 media | Hyclone | SH30027.01 | |
Fetal Bovine Serum | Atlanta Biologicals | S12450H | |
L-glutamine | Corning | MT25005CI | |
HEPES | Hyclone | SH30237.01 | |
Cytation 3 plate reader | Biotek | Interchangable with any fluorescent plate reader and microscope | |
Gen5 Software | Biotek | Recording and analysis of rezasurin coversion | |
Rifampicin | Fisher Scientific | BP2679250 | Prepare 10 mg/ml stock solution in H2O |
Moxifloxacin Hydrochloride | Acros Organics | 457960010 | Prepare 10 mg/ml stock solution in H2O |
Resazurin Sodium Salt | Sigma | R7017 | Prepare 800 μg/mL stock solution in H2O; filter sterilize |
Tween-80 | Fisher Scientific | T164500 | Prepare 20% stock solution in H2O; filter sterilize |